| Popis: |
Somites are precursors of the vertebrae, ribs, and associated musculature and their formation relies on ordered and timely segmentation during early embryonic development. Somites form as they ‘pinch’ away from the pre-somitic mesoderm (PSM) with a species-specific periodicity, coinciding with waves of cycling mRNA expression of ‘clock’ genes sweeping across the PSM that are regulated by a molecular oscillator. Mathematical models suggest that sustained rapid oscillations of Notch-regulated gene transcription in the PSM depend on transient negative feedback loops that rely on unstable protein products. However, the pacemaker mechanism that underlies this segmentation clock is poorly understood and little experimental evidence exists linking the level of clock proteins to the periodicity of clock gene oscillations. It was previously shown that pharmacological inhibition of Wnt signalling slows oscillating transcription of the Notch target Lfng in the PSM of the chicken and mouse embryos. We have shown that another Wnt inhibitor XAV939 and a number of cdk inhibitors can phenocopy this effect in the PSM. This effect appears independent of the cell cycle and these inhibitors appear to have a general effect on transcription in the chicken PSM. In contrast to a previous report, we find that direct inhibition of Sonic hedgehog (Shh) signalling has no effect on oscillating clock gene transcription in the chicken PSM. A custom-made antibody reveals that the level of a key clock protein is increased in the chicken PSM when treated with XAV939, or either of the cdk inhibitors which also slow oscillating clock gene transcription. This molecular evidence supports models which predict that the level of clock proteins in the PSM is fundamental to maintain rapid clock oscillations and timely somite formation. The Microarray and RNA-seq analyses of chicken PSM tissues discovered a set of genes whose transcription is affected by all three inhibitor treatments relative to corresponding controls. These candidates will be studied further as potential regulators of the segmentation clock period. Immunoprecipitation with the custom-made antibody followed by mass spectrometry analysis of lysates from chicken PSM tissues treated with reagents that modify clock pace will uncover any post-translational modifications of this key protein which are altered by these inhibitors with the aim of identifying any key enzymatic regulators of its stability which act as part of the segmentation clock pacemaker mechanism. |
